Novel membrane sensor for determination of lamotrigine in pharmaceuticals and urine

Lamotrigine (LMT), chemically known as [6-(2,3-dichlorophenyl)-1,2,4-triazine-3,5-diamine], is a broad spectrum antiepileptic drug, used as monotherapy and as an adjunct with other antiepileptic drugs for treatment of partial and generalized toxic-clonic seizures.

* Corresponding author Tel: +91 9880547493

E-mail address: prasadtnpur@gmail.com (N Rajendraprasad)

© 2019 by the authors; licensee Growing Science, Canada

doi: 10.5267/j.ccl.2019.002.002

Current Chemistry Letters 8 (2019) 87–96

Contents lists available at GrowingScience

Current Chemistry Letters

homepage: www.GrowingScience.com

Novel membrane sensor for determination of lamotrigine in pharmaceuticals and urine

N Rajendraprasad a*

a PG Department of Chemistry, JSS College of Arts, Commerce and Science (Autonomous under University of Mysore), B N Road, Mysuru-570 025, Karnataka, India

C H R O N I C L E A B S T R A C T

Article history:

Received July 28, 2018

Received in revised form

February 20, 2019

Accepted February 20, 2019

Available online

February 22, 2019

Lamotrigine (LMT), chemically known as [6-(2,3-dichlorophenyl)-1,2,4-triazine-3,5-diamine], is a broad spectrum antiepileptic drug, used as monotherapy and as an adjunct with other antiepileptic drugs for treatment of partial and generalized toxic-clonic seizures It is used to treat neurological lesions and as a tranquilizer A selective electrochemical membrane sensor has been developed and validated for determination of LMT The membrane constructed using LMT and molybdophosphoric acid in THF and PVC is applicable for the detection of 5 × 10 -4 to 9 × 10 -3 M LMT in the pH range between 4.6 and 5.8 with the Nernstian slope of 57.14±1 mV/decade The regression coefficient value of 0.9932 showed a good linear correlation between the concentrations of LMT and measured cell potentials The limits of detection (LOD) and quantification (LOQ) values for the fabricated sensor were 1.3 × 10 -5 and

4 × 10 -5 M LMT, respectively Various experimental conditions were optimized to reach the effective performance characteristics of the sensor The effect of various cations, anions and organic species on the performance of sensor was studied by following standard-addition procedure The results revealed no such variations due to presence of foreign ions or species The fabricated sensor was subjected to validation to check accuracy, precision, robustness and ruggedness The mean accuracy for determination of LMT was found to be 99.16% The developed sensor was successfully used to determine LMT in tablets and in spiked human urine

© 2019 by the authors; licensee Growing Science, Canada

Keywords:

Membrane sensor

Lamotrigine

Potentiometric determination

Pharmaceuticals

Spiked human urine

1 Introduction

Lamotrigine (LMT) is chemically known as 3,5-diamino-6-(2,3-dichlorophenyl)-as-triazine (Fig 1), is an anticonvulsant drug used in the treatment of epilepsy and bipolar disorder It is also used

off-label as an adjunct in treating clinical depression

Fig 1 Chemical structure of LMT

As an antiepileptic drug LMT is attracted by many analysts The monograph in the United States

monobasic potassium phosphate buffer, triethylamine and acetonitrile as mobile phase The drug LMT

Various methods have been reported for its determination in pharmaceuticals and they are titrimetry

electrodes are based on PVC membranes doped with tetraphenyl borate (TPB) or LMT-phosphotungstic acid (PT) ion-pair complexes as molecular recognition materials The electrodes are used for determination of LMT in urine and plasma There is also a report for LMT determination using

determine LMT, they failed to present outcomes of detailed validation aspects

Research in the field of development of potentiometric sensors is gaining more and more attention and a number of potentiometric sensors have been developed for the determination of species in the

applications to quantify the compounds since they neither require sophisticated instrument nor relying

on stringent experimental conditions Therefore, an attempt is made to develop a novel potentiometric membrane sensor for the determination of LMT in pharmaceuticals and spiked human urine The membrane sensor is fabricated by preparing the ion pair complex of LMT with molybdophosphoric acid and its membrane with polyvinyl chloride in THF Different parameters are optimized to improve the selectivity of membrane for accurate and precise determination of LMT The fabricated sensor is used to determine LMT in pharmaceuticals and spiked human urine

2 Experimental

2.1 Apparatus

copper wire were used for potential measurements

2.2 Materials and methods

The chemicals and reagents used were of analytical grade Distilled water was used throughout the work The pure LMT (99.8%) was kindly provided by Torrent Pharmaceuticals Ltd (Mumbai, India) Lamitor-DT tablets (100mg LMT/tablet) (Indrad-382721, Mehsana, India) were purchased from local commercial sources Dodeca-molybdophosphoric acid (PMA), tetrahydrofuran (THF) and polyvinyl chloride (PVC) were supplied by S D Fine Chem Ltd, Mumbai, India Concentrated sulphuric acid

from a 21 year male healthy volunteer, it was filtered and diluted ten times with water before use

water A 0.01 M PMA solution was prepared by dissolving calculated amount the compound in distilled

sucrose, fructose, glucose, maltose, starch, lactose, glycine, sodium fluoride, calcium chloride, nickel chloride, potassium chloride, ammonium chloride, cadmium chloride and cobalt chloride were prepared

by dissolving required weight of the respective compound (all from S.D Fine Chem Ltd., Mumbai, India) in distilled water

2.3 Preparation standard LMT solution

A standard solution of 0.01M LMT was prepared by accurately dissolving calculated quantity of

2.4 PROCEDURE

2.4.1 Fabrication of the sensor

A mixture of 20 mL of each of 0.01M solutions of LMT and PMA was stirred for 20 minutes and the resulted precipitate was collected on Whatmann No 41 filter paper by filtration The precipitate was dried overnight at room temperature A 20 mg of dried precipitate was taken in a Petri Dish of 4 cm width, about 0.1g of PVC and 10ml of THF were added The content after mixing was allowed to evaporate under room temperature for 24 hours The dried membrane was fused to one end of non-conducting glass tube with the aid of THF The dried tube was filled by 3-5 mL internal solution of 0.01M LMT A pure copper wire of 2.0 mm diameter and 15 cm length was tightly insulated leaving 1.0 cm at one end and 0.5 cm at other end for connection. One terminal of the wire was inserted into internal solution and the other terminal was connected to the potentiometer The sensor was conditioned

by soaking in analyte solution for 6 hours

2.4.2 Preparation of calibration curve

Into a series of 10.0ml volumetric flasks varying aliquots (0.0, 0.5, 1.0, 1.5, 3.0, 4.5, 6.0, 7.5 and 9.0 ml) of 0.01M standard LMT solutions were placed with the help of a microburet The volume of each flask was adjusted to 10 mL with water The potential of each solution was measured by using

LMT-PMA sensor versus Ag/AgCl reference electrode

The calibration graph of measured potential versus –log [LMT] was prepared The concentration

of the unknown was found by using calibration graph or regression equation derived using potential

and –log [LMT] data

2.4.3 Procedure for interference study

In a 10 ml volumetric flask, 2 ml of 0.01M drug solution and 2ml of 1mM solution of interferent were taken The solution after adjusting to pH 5 and diluting to the mark, the potentials of each were measured using the electrochemical cell assembled for preparation of calibration curve

2.4.4 Procedure for tablets

      Twenty tablets were weighed and transferred in to a clean dry mortar and powdered Portion of the tablet powder equivalent to 64.02 mg of LMT was transferred in to a 25 ml volumetric flask and shaken

mixed well and filtered through Whatmann No 41 filter paper A suitable aliquot was taken and its potential measured by following the procedure described for preparation of calibration curve The concentration of LMT was calculated using the calibration curve or regression data

2.4.5 Procedure for spiked human urine

In a 10ml volumetric flask 1ml of 1:10 urine and 2ml of 0.01M LMT solution were taken The volume was brought to the mark with water and mixed well After bringing the solution to the optimum

pH of 5 the potential of the solution was measured using LMT-PMA sensor and Ag-AgCl reference electrode The concentration of LMT in the solution was calculated using the calibration curve or regression data

3 Results and discussion

The development and validation of ion-selective electrodes using membranes is of great interest for pharmaceutical analysis because they offer the advantages of simplicity of fabrication and operation, rapid response time, fair detection limits, acceptable selectivity, accuracy and precision, applicable to the detection of wider concentration range of species in coloured and turbid solutions, and probability

to automate and computerize

The membrane was prepared based on the reaction between aqueous cationic LMT with the solution

of dodeca-molybdophosphate (PMA) to form a stable 1:1 water insoluble ion association complex, with low solubility product and suitable grain size precipitate The probable reaction scheme for the formation of LMT-PMA ion-association complex is given in scheme 1 The formed ion-associate of LMT-PMA was used to fabricate the membrane consisting with poly-vinyl chloride (PVC) using tetrahydrofuran (THF)

Cl

Cl

N

N N

Cl Cl

N

N N

Mo O O HO

- O P

O OH OH

O O HO

- O P

O OH OH

Scheme 1 Reaction pathway for formation of LMT- PMA ion-pair complex

The following systematic representation is depicted for the electrochemical cell assembly:

AgCl Reference electrode║ LMT-PMA Sensor│0.01M LMT solution│Cu-Wire

3.1 Optimization of variables

Different experimental variables such as pH, soaking time, response time, stability and effect of interferents were optimized by measuring the potential of the LMT solution of known concentration using the developed sensor

The optimum pH range of the sensor was found to be from 4.6 to 5.8 and between which the potential measured for each solution of LMT of any concentration within the linear range were almost

constant There were lower potential values observed at pH lesser than 4.6 and 5.8 (Fig 2)

The soaking time was examined by immersing the sensor into a solution of LMT of known concentration for different time periods From a series of investigations it was found that the average

soaking time for the conditioning of the electrode is 6 hours (Fig 3) Therefore, it was found necessary

for the sensor to soak in standard analytic solution at least for 6 hours prior to its use for analyses

Fig 2 Effect of pH on EMF (1.0 × 10-3 M LMT) Fig 3 Effect of soaking time on EMF (1.0 ×

10-3 M LMT)

20

40

60

80

100

120

140

160

180

200

pH

20 40 60 80 100 120 140 160 180 200

Time, h

B

After immersing the LMT-PMA sensor along with the reference electrode into the solution of

analyte or containing analyte reproducible and constant potential readings were observed in less than 5

seconds (Fig 4) Therefore, the response time of the sensor was found as 5 seconds

The developed sensor was subjected to measure the potential of LMT solution in the presence of

various organic and inorganic compounds, cations and anions as interferents The study was undertaken

This confirmed that the sensor is selective for the determination of LMT in the presence of such charged

or neutral species

Fig 4 Study of response time (1.0 × 10-3 M LMT) Fig 5 Calibration graph

3.2 Method validation

The electrochemical response parameters of developed LMT-PMA sensor were evaluated

showed that the sensor provides rapid, stable and linear response for the LMT concentration ranged

of 57.14±1 mV/decade Stable potentiometric readings were obtained with variations within ±1 mV for

the span period of more than a month The lower limit of detection, calculated from the intercept of the

also been presented in Table 1

Table 1 Electrochemical characteristics of the LMT-PMA sensor

Parameters Values

3.2.1 Accuracy and precision

Accuracy and intra- and inter-day precision were evaluated by analysing pure LMT solutions at

three different concentrations in seven replicates during the same day and five replicates during

different days The amount of LMT found was calculated for each measurement The RE (%) and the

RSD (%) values were calculated The percent relative error which is an index of accuracy is ≤4.50 and

150

155

160

165

170

175

180

120 130 140 150 160 170 180 190 200

-log[LMT]

is indicative of acceptable accuracy The obtained RSD values ranged between 2.11 and 4.34%

indicated that the results are prcise enough The results of this study are presented in Table 2

Table 2 Results of accuracy and precision study

LMT

taken,

LMT found,

% RE

%

RSD

LMT found,

%

RE

%

RSD

3.00

6.00

9.00

3.05 5.73 9.24

1.67 4.50 2.67

2.11 2.42 2.39

3.03 5.84 8.76

1.00 2.67 2.67

2.45 4.34 2.36

3.2.2 Robustness and ruggedness

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small,

but deliberate variations in experimental parameters and provides an indication of its reliability for

normal usage Under deliberately varied experimental conditions [pH: 4.6(±2) – 5.8(±2) and

temperature: 25±2 ᴼC] the %RSD values ranged from 1.11 to 2.12% revealed robustness In method

ruggedness, the analyses with different potentiometers, on different days by different analysts were

performed Such variations did not yield any appreciable changes in the measurement The

inter-instrumental and inter-analysts RSD values of <3.4% declared that the proposed potentiometric sensor

is robust enough The results of robustness and ruggedness studies are presented in Table 3

Table 3 Results of method robustness and ruggedness study, expressed as %RSD

LMT

taken,

M

Robustness Ruggedness

3.00

6.00

9.00

2.12 1.11 1.94

1.92 1.99 1.20

1.89 1.62 1.90

1.32 1.87 1.63

3.2.3 Application to tablets

A 5 mL of 0.01M LMT solution of tablets extract prepared under ‘procedure for tablets’ was

subjected to analysis by the optimized procedure The mean measured potential for the tablets extract

was found as same as that obtained for the pure drug solution The results in this study were compared

LMT is quantified using a mixture of monobasic potassium phosphate buffer, triethylamine and

acetonitrile as mobile phase The accuracy and precision were evaluated by applying Student’s t- test

and variance ratio F- test, respectively The calculated t- and F- values at 95% confidence level did not

exceed the tabulated values and this confirmed insignificant difference between the results of reference

and proposed methods The mean percent recovery of LMT from tablets was found to be 98.9 with

RSD value of 3.2% These results are presented in Table 4

Table 4 Results of analysis of tablets by the proposed method and statistical comparison of the

results with the reference method

Tablet

Reference

F = 1.27

3.2.4 Recovery study

A standard addition procedure was followed to further assure the accuracy of the sensor The solutions were prepared by spiking pure drug into a pre-analyzed tablet extract at three different levels and potentials measured using the sensor To a 2 mL of 0.01M LMT tablets extract, 1, 2 and 3 mL of 0.01

M pure LMT drug solutions were spiked (five replicates), and pH was adjusted After diluting the solutions to 10 mL, the potentials of each were measured and the amounts of LMT calculated The recovery of pure LMT was computed The percentage recovery of LMT from tablets, presented in Table

5, ranged from 98.33 to 102.4% revealed that the sensor is selective to give satisfactory in the presence

of excipients

Table 5 Results of accuracy assessment by recovery test for Lamitor-DT tablets

LMT in

tablet,

Pure LMT added,

Total found,

Pure LMT recovered (Percent±SD*) 3.01

3.01

3.01

1.50 3.00 4.50

4.54 5.96 7.62

102.0±1.23 98.33±0.66 102.4±1.18

*Mean value of three measurements

3.2.5 Spiked human urine analysis

From the analysis of spiked human urine sample the percent recovery of LMT were ranged from 93.6 to 98.6% with RSD of <5% indicated that the endogenous substances did not interfere to the assay and hence the sensor is suitable for its use in physiotherapeutic administration of LMT

4 Conclusions

This is the first paper describing the fabrication of membrane sensor using phosphomolybdic acid and its application to determine lamotragine in pharmaceuticals and spiked human urine The sensor provides fast and linear Nernestian response over a wide range of lamotragine concentration The sensor has been successfully used to determine drug content in pure state, tablets and spiked human urine with acceptable recovery The results obtained were accurate and precise with good agreement to consider the sensor for its use as a tool to determine lamotragine in quality control laboratories The electrochemical cell’s assembly is a simple, low cost and selective tool for direct determination of lamotragine in aqueous media without involving any tedious extraction step

Acknowledgement

Author thanks Torrent Pharmaceuticals Ltd., Mumbai, India, for gifting pure lamotrigine sample The author is indebted to UGC, SWRO, Bengaluru, India, for financial assistance in the form of Minor Research Project Grant of Award No 1495-MRP/14-15/KAMY013/UGC-SWRO, dated 04-02-15, to pursue this research work The author is grateful to the Principal of JSS College of Arts, Commerce and Science, B N Road, Mysuru, India, for providing the facilities to pursue this work

References

Convention, USA, 2000

lamotrigine in plasma and tablets by planar chromatography and comparison with liquid

chromatography and UV spectrophotometry J AOAC Intl., 79(6), 1277

Simultaneous liquid chromatographic determination of lamotrigine, oxcarbazepine

monohydroxy derivative and felbamate in plasma of patients with epilepsy J Chromatogr B.,

828(1-2), 113

simple and rapid liquid chromatographic method Ther Drug Monit., 20(2), 209

chromatographic determination of six antiepileptic drugs and two metabolites in microsamples

of human plasma Ther Drug Monit., 21(5), 559

plasma by high-performance liquid chromatography J Chromatogr B., , 817(2), 199-206

chromatographic method for the determination of lamotrigine in plasma J Pharm Biomed

Anal., 17(3), 525

human plasma by high-performance liquid chromatography J Chromatogr B Biomed Sci

Appl., 691(1), 139

determination of lamotrigine, zonisamide, and carbamazepine in human plasma by

high-performance liquid chromatography Biomed Chromatogr., 21(3), 225

10 Patil, K.M., Bodhankar, S.L (2005) Simultaneous determination of lamotrigine, phenobarbitone, carbamazepine and phenytoin in human serum by high-performance liquid

chromatography J Pharm Biomed Anal., 39(1-2), 181

11 Saracino, A.P., Maria, A.M., Koukopoulos, G.M., Sani, M.M.A., Raggi, M.A (2007) Simultaneous high-performance liquid chromatographic determination of olanzapine and

lamotrigine in plasma of bipolar patients Ther Drug Monit., 29(6), 773

12 Bartoli, A., Marchiselli, R., Gatti, G (1997) A rapid and specific assay for the determination

of lamotrigine in human plasma by normal-phase HPLC Ther Drug Monit., 19(1), 100

13 Cociglio, M., Alric, R., Bouvier, O (1991) Performance analysis of a reversed-phase liquid

chromatographic assay of lamotrigine in plasma using solvent-demixing extraction J

Chromatogr.B: Biomed Sci Appl., 110 (1), 269

14 Lensmeyer, G.L., Gidal, B.E., Wiebe, D.A (1997) Optimized high-performance liquid chromatographic method for determination of lamotrigine in serum with concomitant

determination of phenytoin, carbamazepine, and carbamazepine epoxide Ther Drug Monit.,

19(1), 292

15 Angelis-Stoforidis, P., Morgan, D.J., O’Brien, T.J., Vajda, F.J.E (1999) Determination of

lamotrigine in human plasma by high-performance liquid chromatography J Chromatogr B

Biomed Sci Appl., , 727(1), 113

16 Croci, D., Salmaggi, A., de Grazia, U., Bernardi, G (2001) New high-performance liquid

chromatographic method for plasma/serum analysis of lamotrigine Ther Drug Monit., , 23(6),

665

17 Castel-BrancoAlmeida, A.M., Falcao, A.C., Macedo, T.A., Caramona, M.M., Lopez, F.G

(2001) Lamotrigine analysis in blood and brain by high-performance liquid chromatography

J Chromatogr B Biomed Appl., 755(1), 119

18 Rajendra Prasad, N., Basavaiah, K., Vinay, K.B (2009) Determination of drug content of

pharmaceuticals containing Lamotrigine by titrimetry in non-aqueous medium Proc Indian

Nat Sci Acad., 75(3), 131

19 Rajendra Prasad, N., Basavaiah, K., Vinay, K.B (2010) Sensitive spectrophotometric determination of lamotrigine in bulk drug and pharmaceutical formulations using bromocresol

green Ecl Quim., 35(1), 55

20 Rajendra Prasad, N., Basavaiah, K., Vinay, K.B (2010) Micro and nanogram determination of

lamotrigine in pharmaceuticals by visible spectrophotometry using bromophenol blue Indian

J Chem Tech., 17(5), 220

lamotrigine in pharmaceutical preparations and urine by charge-transfer complexation

Pharmazie., 63(11), 791

determination of lamotrigine Indian J Pharm Sci., 62(1), 51

23 Youssef ,N.F., Taha, E.A (2007) Development and validation of spectrophotometric, TLC and

HPLC methods for the determination of lamotrigine in presence of its impurity Chem Pharm

Bull., 55(4), 541

24 Elizabeth, G-S., Giannoutsos, S., Lower, D.R., Virji, M.A., Krasowski, M.D (2007) Drug monitoring: simultaneous analysis of lamotrigine, oxcarbazepine, 10-hydroxycarbazepine, and zonisamide by HPLC-UV and a rapid GC method using a nitrogen-phosphorus detector for

levetiracetam J Chromatogr Sci., 45( 9), 616

25 Anantha Kumar, D., Venkata Kumar, C., Seetharamaiah, P., Sheshagiri Rao, J.V.L.N (2010)

Estimation of lamotrigine by RP-HPLC method J Chem., 7(S1), S203

26 Vinay, K B., Revanasiddappa, H D., Cijo1, M X., Ramesh, J., Swamy, N., Rajendraprasad,

N (2015) Stability indicating ultra performance liquid chromatographic assay of lamotrigine

in pharmaceuticals Trakia J Sci., 13(1), 1

27 Olga, D.R., Calvo, M.E., Acros-Martinez, M.J (2008) Determination of lamotrigine in pharmaceutical preparations by adsorptive stripping voltammetry using screen printed

electrodes Sensors, 8(12), 4201

28 Gupta, V K., Singh, A K., Gupta, B (2007) Development of membrane electrodes for

Bioanal Chem., 389, 2019

29 Hayedeh B.S., Soltan A E., Atefeh T., Fatemeh B., Hadi A., Nasim A., Somayeh M (2011)

Potentiometric Sensing of Lamotrigine Based on Molecularly Imprinted Polymers Electroanal 23(11), 2716

30 Ensafi, A., Allafchian, A R (2008) Potentiometric sensor for betahistine determination in

pharmaceuticals, urine and blood serum J Pharm Biomed Anal., 47(4-5), 802

31 Kanberoglu, G S., Coldur, F., Topcu, C., Cubuk, O (2015) PVC-Membrane Potentiometric

Sensor for the Determination of Tamoxifen in Pharmaceutical Formulations IEEE Sensors

J., 15(11), 6199

32 AlRabiah, H., Al-Majed, A., Abounassif, M., Mostaf, G.A.E (2016) Two novel potentiometric

sensors for determination of clonidine in some pharmaceutical formulation Int J Electrochem

Sci., 11, 6761

33 Ganjali, M.R., Karimi, S., Shahtaheri, S.J., Norouzi, (2013) Determination of clonidine by

potentiometry using PVC membrane electrode Int J Electrochem Sci., 8, 1999

34 Del Valle, E.M (2004) Cyclodextrins and their uses: a review Process Biochem., 39, 1033

35 Hedges, A.R (1998) Industrial Applications of Cyclodextrins Chem Rev., 98, 2035

36 Yang, R., Li, K.A., Wang, K., Zhao, F., Li, N., Liu, F (2003) Porphyrin assembly on β-cyclodextrin for selective sensing and detection of a zinc ion based on the dual emission

fluorescence ratio Anal Chem., 75, 612

37 El-Kosasy, A.M., Nebsen, M., El-Rahman, M.K.A., Salem, M.Y., El-Bardicy, M.G (2011) Comparative study of 2-hydroxy propyl beta cyclodextrin and calixarene as ionophores in

potentiometric ion-selective electrodes for neostigmine bromide Talanta, 85, 913

38 Trojanowicz, M (2014) Enantioselective electrochemical sensors and biosensors: A

mini-review Electrochem Comm., 38, 47

39 Patil, S.R., Turmine, M., Peyre, V., Durand, G., Pucci, B (2007) Study of β-cyclodextrin/fluorinated trimethyl ammonium bromide surfactant inclusion complex by

fluorinated surfactant ion selective electrode Talanta, 74, 72

40 El-Kosasy, A.M (2003) Determination of hydroxyurea in capsules and biological fluids by

ion-selective potentiometry and fluorimetry J AOAC Intl., 86, 15

41 Ozoemena, K.I., Stefan, R-I (2005) Enantioselective potentiometric electrodes based on

alpha-, beta-alpha-, and gamma-cyclodextrins as chiral selectors for the assay of l-proline Talantaalpha-, 66alpha-, 501

42 IUPAC Analytical Chemistry Division, Recommendation for Nomenclature of Ion Selective

Electrode Pure Appl Chem., 1994, 66, 2527

43 IUPAC Analytical Chemistry Division, Potentiometric selectivity coefficients of ion selective

electrodes Pure Appl Chem., 2000, 72, 1851

© 2019 by the authors; licensee Growing Science, Canada This is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/)